Genomic characterization of Salmonella isolated from retail chicken and humans with diarrhea in Qingdao, China

Salmonella, especially antimicrobial resistant strains, remains one of the leading causes of foodborne bacterial disease. Retail chicken is a major source of human salmonellosis. Here, we investigated the prevalence, antimicrobial resistance (AMR), and genomic characteristics of Salmonella in 88 out of 360 (24.4%) chilled chicken carcasses, together with 86 Salmonella from humans with diarrhea in Qingdao, China in 2020. The most common serotypes were Enteritidis and Typhimurium (including the serotype I 4,[5],12:i:-) among Salmonella from both chicken and humans. The sequence types were consistent with serotypes, with ST11, ST34 and ST19 the most dominantly identified. Resistance to nalidixic acid, ampicillin, tetracycline and chloramphenicol were the top four detected in Salmonella from both chicken and human sources. High multi-drug resistance (MDR) and resistance to third-generation cephalosporins resistance were found in Salmonella from chicken (53.4%) and humans (75.6%). In total, 149 of 174 (85.6%) Salmonella isolates could be categorized into 60 known SNP clusters, with 8 SNP clusters detected in both sources. Furthermore, high prevalence of plasmid replicons and prophages were observed among the studied isolates. A total of 79 antimicrobial resistant genes (ARGs) were found, with aac(6′)-Iaa, blaTEM-1B, tet(A), aph(6)-Id, aph(3″)-Ib, sul2, floR and qnrS1 being the dominant ARGs. Moreover, nine CTX-M-type ESBL genes and the genes blaNMD-1, mcr-1.1, and mcr-9.1 were detected. The high incidence of MDR Salmonella, especially possessing lots of mobile genetic elements (MGEs) in this study posed a severe risk to food safety and public health, highlighting the importance of improving food hygiene measures to reduce the contamination and transmission of this bacterium. Overall, it is essential to continue monitoring the Salmonella serotypes, implement the necessary prevention and strategic control plans, and conduct an epidemiological surveillance system based on whole-genome sequencing.

Salmonella, especially antimicrobial resistant strains, remains one of the leading causes of foodborne bacterial disease.Retail chicken is a major source of human salmonellosis.Here, we investigated the prevalence, antimicrobial resistance (AMR), and genomic characteristics of Salmonella in 88 out of 360 (24.4%) chilled chicken carcasses, together with 86 Salmonella from humans with diarrhea in Qingdao, China in 2020.The most common serotypes were Enteritidis and Typhimurium (including the serotype I 4, [5],12:i:-) among Salmonella from both chicken and humans.The sequence types were consistent with serotypes, with ST11, ST34 and ST19 the most dominantly identified.Resistance to nalidixic acid, ampicillin, tetracycline and chloramphenicol were the top four detected in Salmonella from both chicken and human sources.High multi-drug resistance (MDR) and resistance to third-generation cephalosporins resistance were found in Salmonella from chicken (53.4%) and humans (75.6%).In total, 149 of 174 (85.6%)Salmonella isolates could be categorized into 60 known SNP clusters, with 8 SNP clusters detected in both sources.Furthermore, high prevalence of plasmid replicons and prophages were observed among the studied isolates.A total of 79 antimicrobial resistant genes (ARGs) were found, with aac(6′)-Iaa, bla TEM-1B , tet(A), aph(6)-Id, aph(3″)-Ib, sul2, floR and qnrS1 being the dominant ARGs.Moreover, nine CTX-M-type ESBL genes and the genes bla NMD-1 , mcr-1.1, and mcr-9.1 were detected.The high incidence of MDR Salmonella, especially possessing lots of mobile genetic elements (MGEs) in this study posed a severe risk to food safety and public health, highlighting the importance of improving food hygiene measures to reduce the contamination and transmission of this bacterium.Overall, it is essential to continue monitoring the Salmonella serotypes, implement the necessary prevention and strategic control plans, and conduct an epidemiological surveillance system based on whole-genome sequencing.

Introduction
Salmonella is one of the common pathogens causing sporadic cases or outbreaks of gastroenteritis (Jain et al., 2020).In 2010, there were an estimated 153 million cases of non-typhoidal Salmonella enteric infections worldwide, of which about 50% were foodborne (Kirk et al., 2015;WHO, 2022).In the United States, foodborne salmonellosis causes an estimated 212,500 infections and 90 deaths annually (CDC, 2019).In China, Salmonella is the second most common bacteria causing foodborne outbreaks (Sun et al., 2021).In fact, consuming contaminated foods, especially poultry meat, directly threatens human health, which appears to be one of the major sources of human infection (Heredia and García, 2018;Pan et al., 2019;Yang et al., 2020).Poultry meat in the food production supply chain have been frequently associated with human salmonellosis cases and are an important cause of Salmonella transmission between poultry farms and humans (Antunes et al., 2016;Rincón-Gamboa et al., 2021).Furthermore, Salmonella is also a significant repository of antimicrobial resistance genes, posing substantial challenges to public health and security (Jajere, 2019).
Over recent years, resistance to cephalosporins have been increasingly reported in humans and poultry industry and the contribution of poultry products to the dissemination of extendedspectrum β-lactamase (ESBL) producing Salmonella strains, and the associated dangers for human health, is well documented in certain countries (Antunes et al., 2016).Of concern is the increased incidence of infections caused by ESBL-producing organisms, including Salmonella, because they are resistant not only to most of the β-lactam antimicrobials but also to other antimicrobial classes, leaving few treatment options and the potential for clinical outcomes worse than those of infections caused by non-ESBLproducing strains (Wei et al., 2019;Wang et al., 2020a).Meanwhile, carbapenems and colistin are often used as the last-line treatment for infections caused by multidrug-resistant (MDR), especially ESBL-producing gram-negative bacteria.However, selective pressure from the overuse or misuse of both antimicrobials has resulted in the emergence of carbapenem-and colistin-resistant Enterobacteriaceae (CRE) (Nang et al., 2019;Octavia et al., 2020).Regarding Salmonella isolates, recent studies have demonstrated their resistance to these critical antimicrobial drugs (Wang et al., 2017;Fan et al., 2022;Zhang et al., 2022b).
The New Delhi metallo (NDM)-β-lactamases that can hydrolyze almost all β-lactam antibiotics, have become one of the most commonly reported carbapenemase resistance mechanisms worldwide (Poirel et al., 2010).According to the Comprehensive Antibiotic Resistance Database (CARD 1 ), 43 variants of NDM genes have been identified in 2022, with NDM-1 and NDM-5 being particularly widespread among Enterobacteriaceae including Salmonella (Iovleva and Doi, 2017).Colistin resistance in bacteria has become another significant threat to food safety and public health, and its development was mainly attributed to the plasmid-mediated mcr genes.Currently, a variety of mcr genes, including mcr-1 to -10 have been reported with mcr-9.1 being 1 https://card.mcmaster.ca/homeprevalent in Salmonella (Ling et al., 2020;Hussein et al., 2021).Hence, the increasing antimicrobial resistance in Salmonella needs to be monitored.
Our previous investigation has described the emergence of the bla NDM-1 gene in Salmonella recovered from chicken carcass in slaughterhouse, while the mcr genes were also detected from living chicken in Qingdao (Wang et al., 2017;Zou et al., 2021).However, there is limited available information on the surveillance and genomics studies of Salmonella in retail chicken in Qingdao.In the current study, we therefore investigated the prevalence, AMR, and genomic characteristics of Salmonella from chilled chicken carcasses and humans with diarrhea in Qingdao.Specially, we identified four Salmonella isolates carrying the plasmid-borne mcr-1.1,mcr-9.1, and NDM-1 genes in this study.

Bacterial isolation
A total of 360 chilled chicken carcasses were collected from local markets in Qingdao, China in 2020.All samples were subjected to qualitative analysis for Salmonella using an enrichment method described by the National Food Safety Standard of China-Food microbiological examination, Salmonella (GB 4789.4-2016).Finally, presumptive Salmonella was selected for biochemical confirmation using API 20E test identification test strips (bioMérieux, Marcy l′ Etoile, France), as well as for molecular identification using PCR assay targeting the invA gene (Ed-Dra et al., 2018).In addition, 86 Salmonella isolates from humans with diarrhea collected from enteric clinic settings in the study year in Qingdao, were also included in the study (Supplementary Table S1).The isolates were serotyped using O-and H-antigens by slide agglutination with hyperimmune sera, and the serotypes of Salmonella samples were identified following the Kauffmann-White scheme (Grimont and Weill, 2007).All confirmed Salmonella isolates were stored in brain heart infusion broth with 40% glycerol (Land Bridge, Beijing, China) at −80°C.One isolate was retained from each sample.All the procedures performed in studies involving human participants were in accordance with the ethical standards of the Research Ethics Committee of China National Center of Food Safety Risk Assessment, Beijing, China (approval no.2014003).
Isolates showed resistance to all tested antimicrobials in this study were defined as pan-resistance, while isolates showed resistance to at least three classes of antimicrobials were defined as MDR.

Statistical analysis
The Chi-square test was performed to analyze differences between groups.Data analysis was performed using SPSS 20.0 (SPSS, Chicago, United States).All statistical tests were two-sided; p < 0.05 were considered statistically significant.

Data availability statement
The sequences obtained in this study have been deposited in the NGDC Genome Sequence Archive 6 under accession number CRA012442.
S. Typhimurium/S.I 4,[5],12:i:-displayed the highest diversity with 15 detected SNP clusters, followed by S. Derby with 8, S. Thompson with 6, and S. Enteritidis with 5 (Figure 3A).Among the designated SNP clusters (n = 35) of the chicken isolates, 21 of them had a minimum SNP distance within environmental source below 21 (Figure 3B), while 14 of them had a minimum SNP distance within human source below 21 (Figure 3C).In contrast, 24 of the designated SNP clusters (n = 33) of the human-associated isolates had a minimum SNP distance within human source below 21 (Figure 3B), while 15 clusters had a minimum SNP distance within environmental source below 21 (Figure 3C).These findings demonstrated that a large number of the designated SNP clusters contained isolates circulating in both human and environmental sources (Pightling et al., 2018).For the 25 isolates that did not fit into designated SNP clusters, 15 were from retail chicken and 10 were from humans.The most common serotypes among these were S. Typhimurium, S. Enteritidis, and S. Agona.

ARG profiles
To determine the presence of resistance genes, all 174 genome sequences were screened against the ResFinder database.A total of 79 different antibiotic resistance genes (ARGs) were identified in the isolates (Figure 6A).The majority of these ARGs belonged to betalactam resistance (n = 19) and aminoglycoside resistance (n = 19) categories.Among the CTX-M-type beta-lactamases, 9 different bla CTX-M genes were detected, with bla CTX-M-55 (n = 11), bla CTX-M-65 (n = 10), and bla CTX-M-14 (n = 6) being the most prevalent (Figure 6A).
It is worth noting that most of the ARGs were detected on plasmid sequences (Supplementary Figure S1A).The aminoglycoside resistance gene aac(6′)-Iaa presented in all the isolates was found located on chromosome sequences (Supplementary Figure S1B).Additionally, a total of 40 isolates were found carrying no ARGs other than the aac(6′)-Iaa gene, which belonged to a variety of serotypes such as S. Typhimurium (n = 10), S. Enteritidis (n = 9), S. Mbandaka (n = 5), etc.

Discussion
Salmonella is one of the most widespread pathogenic foodborne bacteria in Chinese food products and retail chicken have been recognized as one of the major sources of human salmonellosis (Paudyal et al., 2018).Moreover, there has been an increase in reports of the dissemination of resistant Salmonella from chicken to humans through the chicken production chain (Kongsanan et al., 2021;Manzari et al., 2022).In this study, we surveyed the prevalence, serovar distribution, MDR, and genomic characteristics of Salmonella in chilled chicken carcass and humans with diarrhea sources in Qingdao, China in 2020.
Overall, 24.4% of the retail chilled chicken carcass samples purchased in 2020 in local markets contained Salmonella.This prevalence is lower than what was found in other provinces (average, 39.3%) in China (Wang et al., 2020a), Brazil (31.5%) (Perin et al., 2020), but higher than those in Korea (14.1%) (La et al., 2021), the USA (17.9%) (Peng et al., 2016), and European Union (7.1%) (Gonçalves-Tenório et al., 2018).Thus, chicken meat in retail may be an important source of human salmonellosis in China.Besides, data reported that Salmonella prevalence in chilled poultry meat was statistically higher than that of frozen poultry meat and ambient poultry meat in China (Sun et al., 2021).In China, immersion chilling is employed more frequently and Chinese consumers have a preference for chilled meat (25% market share) over frozen meat (15% market share) (Liu et al., 2017).However, once a sample is contaminated with Salmonella during the immersion process, the contamination may spread among the whole batch of carcasses, leading to an increase in the prevalence of pathogens on finished products (Marmion et al., 2021).Therefore, in response to potential public health pressures, more effective intervention strategies during immersion chilling should be implemented to control the quality and safety of chicken products.
Serotype results suggested a wide range of Salmonella serotypes present in retail chilled chicken carcasses and humans with diarrhea.The dominance of S. Enteritidis and S. Typhimurium among the studied Salmonella isolates, is consistent with the data obtained from chicken and human previously in China and other regions worldwide (EFSA and ECDC, 2018;Balasubramanian et al., 2019;Wang et al., 2020a).Besides, S. Indiana is another frequently detected serotype among chicken samples and is also detected from humans with diarrhea in this study.Accordingly, since 2009, the presence of S. Indiana has seen a remarkable increase, which has become one of the top three common serotypes in China (Zhang et al., 2022a).S. Indiana has been isolated from a wide and diverse variety of sample sources in 18 provinces throughout China and was most frequently recovered from food, followed by animals, the environment and then people, especially children under 6 years old with low immunity (Gong et al., 2017).What is more, Salmonella serovars Enteritidis, Typhimurium, and Indiana are also reported as the most common serotypes associated with human infections and outbreaks (Kuang et al., 2018b;Wu et al., 2018).Thus, the high prevalence of these Salmonella serotypes in chicken carcasses and humans with diarrhea indicates a significant risk to public health.The monitoring of the emergence and prevalence of these Salmonella serotypes is essential for the better control of salmonellosis.
The combination of MLST and serotype detection can facilitate research on the hereditary and evolutionary relationships of Salmonella (Xu et al., 2022).The most common Salmonella sequence types were found to be ST11, ST34, and ST19 in this study, where the corresponding serotypes were S. Enteritidis and S. Typhimurium (including S. I 4,[5],12:i:-).This is consistent with findings from previous reports in China (Wang et al., 2020b;Xu et al., 2022).Accordingly, among S. Enteritidis worldwide, ST11 is the predominant sequence type, accounting for 89% of the sequence types in the EnteroBase database (Ashton et al., 2016).However, a recent report from Malaysia showed that ST1925 was dominantly detected among chicken-associated S. Enteritidis strains (Zakaria et al., 2022).In America, the major prevalent STs are ST34, ST33, and ST11 (Pethplerdprao et al., 2017), which shows that STs may be associated with regions.Furthermore, the other mainly identified sequence types, ST13 (S.Agona), ST17 (S.Indiana), and ST40 (S.Derby), were also previously reported to be associated with salmonellosis in human (Luo et al., 2022;Sun et al., 2022;De Jesus Bertani et al., 2023).Observations from the present study therefore provided important evidence and confirmed further that these types of Salmonella can serve an important role in human diarrhea in Qingdao, China.
Herein, we found that 53.4 and 75.6% of retail chilled chicken carcass-and humans with diarrhea-associated Salmonella isolates were MDR, respectively.High percentages of MDR strains were also observed in Salmonella isolated from poultry (Li et al., 2022), food animals (Tang et al., 2023) and humans (Chen et al., 2022) in various regions of China.The surge in antimicrobial-resistant Salmonella isolates is recognized as a crucial public health issue (Eng et al., 2015).The major of Salmonella isolates among both sources were resistant to four antimicrobials of nalidixic acid, ampicillin, tetracycline and chloramphenicol, which are the first-line drugs used against bacterial infection in animal farms worldwide (Nhung et al., 2016;Lekagul et al., 2019).This finding is consistent with the literature from different countries, including China (Tamang et al., 2011;Cai et al., 2016;EFSA and ECDC, 2018).Our results also suggested that Salmonella isolates in this study showed high level resistance to third generation cephalosporins (ceftazidime and cefotaxime) and quinolone (ciprofloxacin), which are the first-line drugs used against bacterial infection in clinics (Tack et al., 2020).These findings are regarded as significant threats to public health, leading to limited drugs of choice for salmonellosis infection treatment in animals and humans.The high AMR rate of S. Enteritidis, S. Typhimurium (including S. I 4,[5],12:i:-) and S. Indiana, with a widespread AMR spectrum is of concern.Moreover, several Salmonella isolates showed resistance to colistin, which is considered as one of the last-resort therapeutic options for the defense of multidrug-resistant Enterobacteriaceae (Elbediwi et al., 2019).This finding of colistin resistance agreed with results reported by other recent studies in Salmonella isolates from poultry and humans (Fortini et al., 2022;Tang et al., 2022).Of note, two carbapenems (imipenem and meropenem) resistant Salmonella isolates were also found in this study.Therefore, the spread of MDR Salmonella isolates, especially resistant to these clinically important antimicrobials, including fluoroquinolone, third-generation cephalosporins, colistin and carbapenems, will result in challenges with public health.
Phylogenetic analysis revealed a high level of genomic diversity of the Salmonella isolates collected from Qingdao region in China.Eight SNP clusters containing both chicken and human isolated collected in this study were identified through NCBI Pathogen Detection.Six of these clusters (PDS000043691.33,PDS000096798.86,PDS000106143.144,PDS000004748.67,PDS000101103.13,and PDS000026869.266)were featured by involving a large number of strains from different countries, indicating these isolates had been widely spread around the world.In contrast, the other two clusters (PDS000078427.2 and PDS000144678.1)formed distinct, locally restricted clades circulating in a limited number of cities in China.Most of the chicken and human isolates shared no common SNP clusters, indicating multiple origins of these isolates.Interestingly, the major SNP clusters found in the present study comprised numerous strains from clinical infections in China and other countries.This highlights the importance of conducting additional research on the dissemination of strains associated with these SNP clusters.
Mobile genetic elements (MGEs), such as bacteriophages and plasmids, are DNA segments that carry genes involving with DNA movement within genomes (Frost et al., 2005).The presence of intact Salmon 118,970 sal3, Gifsy-2, and Gifsy-1 was found at a higher frequency in the genomes of S. Typhimurium, S. I 4,[5],12:i:-, and S. Enteritidis, whereas such prophage types were seldom observed in other prevalent serotypes such as S. Indiana, S. Agona, and S. Derby.These findings indicate the presence of prophage types is associated with serotypes and may be related to the evolutionary history of different serotypes.Recently, a study of a large-scale investigation on S. Typhimurium has reported the common presence of the abovementioned prophages in S. Typhimurium isolated in China (Wang et al., 2023).Furthermore, the specific phage, Salmon 118,970 sal3 was not only found in Salmonella, but also found in E. coli (Li et al., 2020) and Morganella morganii (Minnullina et al., 2019), suggesting that this phage has a relatively wide host range.The presence of Gifsy-1 and Gifsy-2 and their contribution to virulence in S. Typhimurium has been widely reported (Ho and Slauch, 2001).
Plasmids play a crucial role as the primary vehicles for horizontal gene transfer (HGT) in bacteria (De La Cruz and Davies, 2000).Among the isolates, the Col(pHAD28) plasmid replicon was the most common, and the IncF plasmid replicon was the second most common.Interestingly, the IncF is also one of the most prevalent plasmid replicons in Salmonella isolated from food animals in the United States (Mcmillan et al., 2019).The presence of Col(pHAD28) plasmid replicon was observed in a total of 14 serotypes identified in this study, indicating its high occurrence in Salmonella isolated from Qingdao region in China.Continuous monitoring of the spread of this plasmid is essential, as several studies have reported the presence of PMQR genes within this plasmid (Li et al., 2021;Hurtado et al., 2022).Genomic analysis showed that the Salmonella isolates harbored different antimicrobial resistant genes.All of the studied Salmonella isolates harbored the aac(6′)-Iaa gene.However, aac(6′)-Iaa gene and similar genes usually are transcriptionally silent and rarely become transcriptionally active.AAC(6′)-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively (Salipante and Hall, 2003).The mere presence of this gene does not confer aminoglycoside resistance in Salmonella (Magnet et al., 1999;Neuert et al., 2018).Besides, 81.4% (96/118) of the isolates resistant to the aminoglycoside class harbored different aminoglycoside resistance genes in addition to the aac(6′)-Iaa gene.Among diverse mechanisms of aminoglycoside resistance, enzymatic modification is the most prevalent mechanism in pathogenic bacteria, including Salmonella (Ramirez and Tolmasky, 2010;Biswas et al., 2019).Moreover, our study revealed 52/57 (91.2%) of the isolates resistant to the thirdgeneration cephalosporins harbored different beta-lactam resistance genes, which were common detected in S. Enteritidis, S. Typhimurium, S. I 4,[5],12:i:-, and S. Indiana.The bla genes control the resistance to beta-lactam antimicrobials by hydrolyzing the beta-lactam ring, leading to antibiotic inactivation (Jacoby, 2009;Eguale et al., 2017).In this study, the frequently detected bla TEM-1B gene (37.9%,67/174) conferring resistance to ampicillin, is the dominant beta-lactam in most Salmonella serotypes worldwide (Eguale et al., 2017).Meanwhile, nine different CTX-M-type ESBL-producing were found in this study.During the last decade, the most frequently encountered (particularly in areas of Europe and Asia) ESBL genes were those encoding the CTX-M enzyme family, primarily carried by transferable plasmids and transposons (Cantón et al., 2012).The emergence of CTX-M-type ESBL-producing Salmonella has been reported in clinical cases, animals, and food samples worldwide, including China (Brown et al., 2018;Wang et al., 2020a).Five different tet resistance genes were found in 88/174 (50.6%) of the studied isolates, 84 of which were resistant to tetracyclines.Additionally, six plasmid-mediated quinolone resistance (PMQR) genes, with qnrS1 prevalently detected, were identified in among the examined isolates belonging to different serotypes.These critical genes in Salmonella isolates from both chicken and human related samples present a tremendous public health concern.It is essential that the existence of the acquired antimicrobial resistant genes in bacterial genomes does not inevitably confer phenotypic resistance and vice versa.
Through NCBI's Pathogen Detection platform, a total of 60 SNP clusters was categorized using isolates collected in this study (Supplementary Table S4).A SNP cluster is a cluster of isolates where each isolate is less than or equal to 50 SNPs distant from others, which offers higher resolution than serotype and MLST for outbreak surveillance purpose.In the designated SNP clusters, we identified several SNP clusters carrying PMQR genes.Some of these SNP clusters contained a large number of isolates from various isolation locations harboring specific PMQR genes, for example, qnrS1 in PDS000112649.59 of S. Agona, qnrB6 in PDS000102183.75 of S. Corvallis, and qnrS1 in PDS000115660.28 of S. 4,[5],12:i:-; qnr-carrying strains with the same sequence types have been reported to be circulating globally with a high rate of quinolone resistance (Fernández et al., 2018;Zhang et al., 2020;Cadel-Six et al., 2021;Lee et al., 2021;Chen et al., 2023).
On the other hand, there are qnr-carrying isolates collected uniquely or mostly from China in SNP clusters with small numbers of matched isolates such as PDS000078519.2 of S. Derby, PDS000013413.32 of S. Thompson, and PDS000055747.16 of S. Typhimurium, indicating that these strains were circulating locally in specific regions in China and tend to be emerging threat to public health.Consistently, in a recent study investigating ciprofloxacin resistance in Salmonella from humans, food and animals collected in Shanghai, China, qnr-positive isolates were mostly S. Thompson, S. Derby and S. Typhimurium (Kuang et al., 2018a).
Apart from qnr-carrying isolates, this study also detected three mcr-carrying isolates.The isolate harboring mcr-1.1 was found clustered in the SNP cluster PDS000042829.4 of S. I 4,[5],12:i:-, which contained a total of four mcr-1.1-carryingisolates uniquely from China, with one isolated from a diarrheal patient (Sun et al., 2023) and another one isolated from ready-to-eat pork (Wang et al., 2018).The two mcr-9.1-carryingisolates were designated to the SNP cluster PDS000101102.4 of S. Thompson.This SNP cluster had a total of 14 isolates collected from China, of which 11 were mcr-9.1 positive and 9 were qnrB4 positive.Interestingly, there are 8 isolates carrying both mcr-9.1 and qnrB4.A recent study also reported a S. I 4,[5],12:i:isolate carrying both mcr-9.1 and qnrA1 and it was found that these clinically relevant resistance genes were driven by IncHI2-ST1 plasmids (Vázquez et al., 2023).
Additionally, the bla NDM-1 -carrying S. Enteritidis isolate was associated with the SNP cluster PDS000004748.67,which had a total of 258 matched isolates, but no other isolates within this cluster were bla NDM-1 positive, suggesting the bla NDM-1 positive isolate has recently acquired plasmid carrying bla NDM-1 gene.It should be paid great attention to as the NDM-1-harboring plasmids confer resistance to all β-lactams (Huang et al., 2017).
In summary, the frequently detection of Salmonella from retail chilled chicken carcasses in Qingdao, China, highlighted the importance of improving food hygiene measures to reduce the contamination and transmission of this bacterium.We found a considerable diversity of Salmonella serotypes and sequence types among both chicken and human, with MDR, posing a severe risk to food safety and public health.The finding of eight worldwide spread SNP clusters in this study emphasized additional research on the dissemination of strains associated with these SNP clusters.The MGE results showed high prevalence of plasmid replicons and prophages among Salmonella from both sources, and the presence of these MGEs associated with specific serotypes indicated different evolutionary history of these serotypes.Given the high prevalence of CTX-M type ESBL-producing and PMQR genes identified in this study, we strongly recommend that both clinical and veterinary sectors routinely test for AMR when resistance to these antimicrobials is detected in Salmonella.This will enhance monitoring and guide the selection of effective treatments.Overall, it is essential to continue monitoring the Salmonella serotypes, implement the necessary prevention and strategic control plans, and conduct an epidemiological surveillance system based on whole-genome sequencing.

FIGURE 1
FIGURE 1 Distribution of Salmonella isolates.(A) Number of isolates from retail chilled chicken carcasses and humans with diarrhea.(B) Minimum spanning tree of the 174 Salmonella genomes colored by serotype.(C) Minimum spanning tree of the 174 Salmonella genomes colored by ST.The minimum spanning tree was built using cgMLST allelic profiles with the MSTreeV2 algorithm.

FIGURE 2
FIGURE 2 Antimicrobial resistance of 174 Salmonella isolates collected from retail chilled chicken carcasses and humans with diarrhea in this study.(A) Antimicrobial resistance rate of 174 Salmonella isolates to 13 antimicrobials.(B) Number of Salmonella isolates showing resistance to studied antimicrobials.* and ** mean significant difference with the p value less than 0.05 and 0.01, respectively.

FIGURE 4
FIGURE 4 Plasmid replicons in the Salmonella genomes.(A) Core genome phylogeny of the Salmonella genomes and distribution of plasmid replicons.Color of the heatmap indicates the presence of plasmid replicons.(B) Box plot of plasmid replicon counts of the designated SNP clusters of different serotypes.

FIGURE 3
FIGURE 3 Genomic diversity of Salmonella isolates.(A) Heat map of serotype distribution and the designated SNP clusters from different sources.Color indicates the number of isolates.(B) Box plot of minimum SNP distance within same source of the designated SNP clusters of different serotypes.(C) Box plot of minimum SNP distance across different sources of the designated SNP clusters of different serotypes.

FIGURE 5
FIGURE 5 Prophages in the Salmonella genomes.(A) Core genome phylogeny of the Salmonella genomes and distribution of intact prophages.Color of the heatmap indicates the presence of prophages.(B) Box plot of prophage counts of the designated SNP clusters of different serotypes.

FIGURE 6
FIGURE 6 ARGs in the Salmonella genomes.(A) Core genome phylogeny of the Salmonella genomes and distribution of ARGs.Color of the heatmap indicates the presence of ARGs.(B) Box plot of ARG counts of the designated SNP clusters of different serotypes.

TABLE 1 AMR
of the mainly detected Salmonella serotypes, %(n/N).*means significant difference with the p value less than 0.05.